This invention relates to plant calcium/calmodulin-dependent protein kinases, particularly anther-specific calcium/calmodulin-dependent protein kinases.
Calcium and calmodulin regulate diverse cellular processes in plants (Poovaiah and Reddy, CRC Crit. Rev. Plant Sci. 6:47-103, 1987, and CRC Crit. Rev. Plant Sci. 12:185-211, 1993; Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414, 1992; Gilroy and Trewavas, BioEssays 16:677-682, 1994). Transient changes in intracellular Ca2+ concentration can affect a number of physiological processes through the action of calmodulin (CaM), a ubiquitous Ca2+-binding protein. Ca2+/calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of Ca2+-mediated signals (Veluthambi and Poovaiah, Science 223:167-169, 1984; Schulman, Curr. Opin. in Cell. Biol. 5:247-253, 1993). Extracellular and intracellular signals regulate the activity of protein kinases, either directly or through second messengers. These protein kinases in turn regulate the activity of their substrates by phosphorylation (Cohen, Trends Biochem Sci. 17:408-413, 1992; Stone and Walker, Plant Physiol. 108:451-457, 1995).
In animals, Ca2+/calmodulin-dependent protein kinases play a pivotal role in cellular regulation (Colbran and Soderling, Current Topics in Cell. Reg. 31:181-221, 1990; Hanson and Schulman, Annu. Rev. Biochem 61:559-601, 1992; Mayford et al., Cell 81:891-904, 1995). Several types of CaM-dependent protein kinases (CaM kinases, phosphorylase kinase, and myosin light chain kinase) have been well characterized in mammalian systems (Fujisawa, BioEssays 12:27-29, 1990; Colbran and Soderling, Current Topics in Cell. Reg. 31:181-221, 1990; Klee, Neurochem. Res. 16:1059-1065, 1991; Mochizuki et al., J. Biol. Chem. 268:9143-9147, 1993).
Although little is known about Ca2+/calmodulin-dependent protein kinases in plants (Poovaiah et al., in Progress in Plant Growth Regulation, Karssen et al., eds., Dordrecht, The Netherlands: Kluwer Academic Publishers, 1992, pp. 691-702; Watillon et al., Plant Physiol. 101:1381-1384, 1993), Ca2+-dependent, calmodulin-independent protein kinases (CDPKs) have been identified (Harper et al., Science 252:951-954, 1991; Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414, 1992).
Male gametophyte formation in the anther is a complex developmental process involving many cellular events. During microsporogenesis, microsporocytes undergo meiosis to form tetrads of microspores that are surrounded by a callose wall composed of xcex2-1,3-glucan. The callose wall is subsequently degraded by callase, which is secreted by cells of the tapetum (Steiglitz, Dev. Biol. 57:87-97, 1977), a specialized anther tissue that produces a number of proteins and other substrates that aid in pollen development or become a component of the pollen outer wall (Paciani et al., Plant Syst. Evol. 149:155-185, 1985; Bedinger, Plant Cell 4:879-887, 1992; Mariani et al., Nature 347:737-741, 1990). The timing of callase secretion is critical for microspore development. Male sterility has been shown to result from premature or delayed appearance of callase (Worral et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995).
Induction of male sterility in plants can provide significant cost savings in hybrid plant production, enable production of hybrid plants where such production was previously difficult or impossible, and allow the production of plants with reduced pollen formation to reduced the tendency of such plants to elicit allergic reactions or to extend the life of flowers that senesce upon pollination (e.g., orchids).
Several strategies have been developed for the production of male-sterile plants (Goldberg et al., Plant Cell 5:1217-1229, 1993), including: selective destruction of the tapetum by fusing the ribonuclease gene to a tapetum-specific promoter, TA29 (Mariani et al., Nature 347:737-741, 1990); premature dissolution of the callose wall in pollen tetrads by fusing glucanase gene to tapetum-specific A9 or Osg6B promoters (Worrall et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995); antisense inhibition of flavonoid biosynthesis within tapetal cells (Van der Meer et al., Plant Cell 4:253-262, 1992); tapetal-specific expression of the Agrobacterium rhizogenes rolB gene (Spena et al., Theor. Appl. Genet. 84:520-527, 1992); and overexpression of the mitochondrial gene atp9 (Hernould et al., Proc. Natl. Acad. Sci. USA 90:2370-2374, 1993).
Genes encoding plant calcium/calmodulin-dependent protein kinases (CCaMKs) have been cloned and sequenced. Expression of CCaMK genes is highly organ- and developmental stage-specific. When CCaMK antisense constructs were expressed in plants, the plants were rendered male-sterile. The availability of CCaMK cDNA and genomic DNA sequences makes possible the production of a wide variety of male-sterile plants, including monocotyledonous, dicotyledonous, and other plant varieties. CCaMK promoters are also useful for targeted expression of heterologous genes, as is described in greater detail below.
Accordingly, the present invention provides isolated nucleic acids based on the cloned CCaMK sequences. Nucleic acids that include at least 15 contiguous nucleotides of a native lily (SEQ ID NO: 1) or tobacco (SEQ ID NO: 10) CCaMK gene and hybridize specifically to a CCaMK sequence under stringent conditions are useful, for example, as CCaMK-specific probes and primers. CCaMK promoter sequences are useful for the expression of heterologous genes in anthers of transgenic plants in a developmental stage-specific manner.
Isolated CCaMK nucleic acids can be expressed in host cells to produce recombinant CCaMK polypeptide or fragments thereof, which in turn can be used, for example, to raise CCaMK-specific antibodies that are useful for CCaMK immunoassays, for purification of CCaMK polypeptides, and for screening expression libraries to obtain CCaMK homologs from other plant species. The native CCaMK sequence can be altered, e.g., by silent and conservative substitutions, to produce modified forms of CCaMK that preferably retain calcium/calmodulin-dependent protein kinase activity. Alternately, CCaMK polypeptides can be obtained from plant tissue by standard protein purification techniques, including the use of CCaMK-specific antibodies.
The foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawings.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids.
SEQ ID NO: 1 shows the cDNA sequence of Lilium longiflorum CCaMK.
SEQ ID NO: 2 shows the amino acid sequence of rat hippocalcin.
SEQ ID NO: 3 shows the amino acid sequence of a rat neural visinin-like protein.
SEQ ID NO: 4 shows the amino acid sequence of bovine neurocalcin.
SEQ ID NO: 5 shows the amino acid sequence of a rat neural visinin-like protein.
SEQ ID NO: 6 shows the amino acid sequence of a chicken visinin-like protein.
SEQ ID NO: 7 shows the amino acid sequence of a rat neural visinin-like protein.
SEQ ID NO: 8 shows the amino acid sequence of Drosophila frequenin.
SEQ ID NO: 9 shows the amino acid sequence of the xcex1 subunit of mammalian calmodulin kinase II.
SEQ ID NO: 10 shows the cDNA sequence of Nicotiana tabacum CCaMK.
SEQ ID NO: 11 shows the 1720 base pair Nicotiana tabacum 5xe2x80x2 promoter region.
SEQ ID NOs: 12 and 13 show highly conserved regions of mammalian Ca2+/calmodulin-dependent protein kinase.
SEQ ID NO: 14 shows the amino acid sequence of the GS peptide.
SEQ ID NO: 15 shows the amino acid sequence of the MBP peptide.
SEQ ID NOs: 16-19 show primers used for site-directed mutagenesis of the visinin-like domain of Lilium longiflorum CCaMK.
SEQ ID NO: 20 shows the amino acid sequence of Lilium longiflorum CCaMK.
SEQ ID NO: 21 shows the amino acid sequence of Nicotiana tabacum CCaMK.
SEQ ID NOs: 22-25 show Lilium longiflorum inhibitory peptides.
SEQ ID NOs: 26-28 show the amino acid sequences of the three EF-hand motifs in the visinin-like domain of Lilium longiflorum CCaMK.